The aim of this study was to investigate the probable mode of action of ethanolic extract of Phyllanthus amarus traditionally used to correct erectile dysfunction by monitoring the effect on the concentration of penile cyclic guanosine mono-phosphate (cGMP) and the concentration of plasma Nitric Oxide (NO) in male guinea pigs that were administered different doses (100, 200 and 400 mg kg-1 body weight) of the plant extract using direct immunoassay kits. Physiological saline and sildenafil were given to animals that served as the controls. Phytochemical screening of the plant determined included alkaloids, flavonoids, terpenoids, saponins, tannins, steroids and cardiac glycosides. There was no statistical significant difference in the weight of the animals before and after the administration of the plant extract. The penile weight per body weight ratio showed a significant difference between groups (p<0.05). There was a dose dependent increase in the level of cGMP in the different groups administered different doses of the extract, with the 400 mg kg-1 group showing significant difference compared to lower-dose and saline groups (p<0.05). Plasma nitric oxide concentration was not significantly different among the control and experimental groups. Conclusively, ethanol extract of the plant had a dose dependent increase on the level of cGMP which might account for the claim of the plant been use as a sexual stimulant and the increase level of the cGMP probably had a feedback effect on nitric oxide synthase which resulted to reduced concentration of NO in the plasma.

Sexual function is an important component of quality of life and subjective
well being in humans. Sexual problems are widespread and adversely affect mood,
well being and interpersonal functioning (Laumann et
al., 1999). The main sexual problems are related to sexual desire and
male sexual dysfunction. Successful treatment of sexual dysfunction may improve
not only sexual relationship but also quality of life.

Ginseng, for example, is an essential constituent in traditional Chinese medicine
(Kim et al., 1976) and at least 6 million Americans
use the root of this slow-growing perennial plant (Nocerino
et al., 2000). Phyllanthus amarus is one of such plants claimed
to possess aphrodisiac property. P. amarus (Euphorbiacae) is a small
animal herb that grows to a height of about 6-15 inches found throughout the
tropics and subtropics. It is a broad-spectrum medicinal plant that has received
worldwide recognition. It has recently attained the status of miracle plant
because of its ability to cure several ailments as claimed by its proponents
(Srividya and Periwal, 1995). It is locally called Iyin-olobe
(Yoruba, south- west Nigeria) or kidney stone plant (Adeneye
et al., 2006). In traditional medicine, it is known for its hepatoprotective,
antidiabetic, antihypertensive, analgesic, anti-inflammatory and antimicrobial
properties (Adeneye et al., 2006). The plant
is also used in the treatment of stomach disorders, skin diseases and cold (Iwu,
1993). It has antidiarrheal effect (Odetola and Akojenu,
2000). Its anti- viral activity against hepatitis B virus (Thyagarajan
et al., 1988), anticarcinogenic and antimutagenic activities ( Sripanidkulchai
et al., 2002) anti- nociceptive and anti- inflammatory activities
(Kassuya et al., 2006) have also been reported.
The recognized active lignan in Phyllanthus amarus as reported by Sharma
et al. (1993) are Phyllanthin and Hypophyllanthin and they were reported
to exhibit anti-hepatotoxic property. These lignans comprises of lots of benzene
rings and are bulky, showing similarities with known phosphodiestrase 5 (PDE
5) inhibitors, sildenafil, tadanafil, zaprinast and verdanafil. This might be
an indication that the ethanol extract of Phyllanthus amarus is probably
a PDE5 inhibitor.

Erectile dysfunction is defined as the inability to attain or maintain penile
erection sufficient for sexual performance on at least two-thirds of occasions
(Morales, 2003). An erect penis has always been a symbol
of virility and fertility. The inability to obtain or maintain an erection sufficient
for vaginal penetration is clinically known as erectile dysfunction and or impotence,
which is a health problem of major concern among men from all geographical locations
(Goldstein, 2000). Erection occurs when the smooth muscle
of the corpora cavernosum relaxes, allowing pooling of blood. The relaxation
of the smooth muscle is as a result of the autonomic nervous system through
the action of Cyclic Guanosine Mono-Phosphate (cGMP) acting as a second messenger
as a result of Nitric Oxide (NO) activating guanylyl cyclase. Guanylyl cyclase
brings about increase in production of cGMP by converting Guanyl Triphosphate
(GTP) to cGMP, while phosphodiesterase breaks it down (i.e., cGMP) to Guanosine
Monophosphate (GMP). The most common treatment for erectile dysfunction is a
phosphodiesterase-5 inhibitor. This works by blocking the breakdown of cyclic
GMP that has been generated by nitric oxide. There are 3 phosphodiesterase inhibitors
in general use viz; sildenafil, vardenafil and tadalafil. They prevent the breakdown
of cGMP by inhibiting the action of phosphodiesterase and have gained worldwide
fame for the treatment of male impotency (Goldstein, 2000).

The search for an effective, safe and easy to administer drug for use in erectile
dysfunction, impotence and fertility has been a perennial pursuit of most societies
from times immemorial throughout history. Treatments for these disorders have
been the use of ginseng, rhinoceros horn and other dubious herbs to enhance
potency. The discovery of testosterone in the late 1930s and its use to relieve
the impotence in hypogonadal men was a major step forward in this field. Many
substances used for recreational purposes (or sometimes abused) were also thought
to have profound effects on sexual performance (Morton,
1992). A wide variety of drugs have been reported to possess erctolytic
effects (Meinhardt et al., 1997). L-arginine
(nutrient) and Yohimbe, Panax ginseng, Ginko biloba and maca root (botanicals)
are some products claimed to provide some benefits in correcting sexual dysfunction.
Though little scientific reports are available on the ability of Phyllanthus
amarus to serve as an aphrodisiac. Obianime and Uche
(2009) reported the use of the aerial part of this plant in improving libido
and reproductive function in men, thus supporting the claims of traditional
medicine practitioner of the plant being used as an aphrodisiac.

Impotence is not a disease but a secondary condition brought on by other, primary
causes. These primary causes are mostly either due to the inhibition of the
synthesis of Nitric Oxide (NO), or the fast action of phosphodiesterase (V)
which breaks down cGMP (Merck, 1996). Since P. amarus
is acclaimed to be used as an aphrodisiac there is the need to provide scientific
information on its androgenic potentials and at the same time look at the possible
mode of action of the plant extract. This could be done by determining its effect
on nitric oxide synthesis and on phosphodiesterase activity via cGMP concentration.

The aim of this research was to determine the effect of ethanolic extract of Phyllanthus amarus on plasma Nitric Oxide (NO) and the concentration of cGMP in corpus cavernosum.

MATERIALS AND METHODS

Fresh Phyllanthus amarus plants were collected in August 2009 within the premises of the College of Medicine, University of Lagos Idi-araba. The plant was identified and authenticated at the Department of Botany, Faculty of Sciences, University of Lagos, Akoka, Lagos State. The whole plant was washed and oven dried at 40°C. The dried plant was pulverized with an electric blender (Blender/Miller III model MS-223, China). The powdered plant was then extracted with 96% (v/v) ethanol, by soaking 100 g of dried plant in 1 L of ethanol for 48 h at room temperature with constant shaking. The suspension was filtered with muslin cloth and resulting filtrate was then concentrated using a rotary evaporator (Stuart, RE300). The extract was finally concentrated in a thermo regulated water bath at 20°C for 24 h.

The concentrate was reconstituted in physiological saline to give different required doses of 100, 200 and 400 mg kg-1 body weight. The reconstituted extract was then administered to all animals in different groups.

Phytochemical screening: Phytochemical tests were carried out on the
ground plant using standard procedures to identify the constituents as described
by Sofowara (1993).

Animal grouping and extract administration: A total of thirty (30) male Guinea pigs weighing between 220 and 600 g were obtained from University of Agriculture Abeokuta, Ogun State, Nigeria and were allowed to acclimatise for 30 days before the experiment. They were housed in aerated plastic cages of five animals per cage and were adequately fed normal animal chow throughout the experiment ad libitum. At the end of the acclimatisation period, only the twenty-five of the twenty-seven that survived were used.

The animals were randomly assigned into different groups; A, B, C, D and E
of five animals each. Physiological saline, commercially acquired Sildenafil
Citrate (HAB Pharmaceuticals and Research Ltd) which serve as controls and the
reconstituted extract were administered orally using a rubber dropper as follow:

Group 1:

Control (1mL physiological saline).

Group 2:

1 mL of sildenafil citrate corresponding to 100 mg kg-1 body
weight.

Group 3:

1 mL of the extract corresponding to 100 mg kg-1 body weight.

Group 4:

1 mL of the extract corresponding to 200 mg kg-1 body weight.

Group 5:

1 mL of the extract corresponding to 400 mg kg-1 body weight.

Collection of samples: The animals were anaesthetized by injecting 5
mg kg-1 body weight ketamin intraperitorially. The animals were quickly
dissected and cardiac blood was collected into a heparinized bottle. The penile
tissue was harvested and immediately frozen in liquid nitrogen and stored at
-20°C pending cGMP analysis. The blood was transferred into centrifuge bottles
and centrifuged at 400 x g for 15 min to get the plasma which was used for nitric
oxide assay.

Determination of penile tissue (corpus cavernosum) cGMP: Frozen penile tissue samples were homogenized placed on an ice bath with 5 mL of 0.1 M HCl in a glass mortar. Centrifugation was done at 5,000 rpm for 10 min at room temperature and the supernatant was collected for quantitative immunoassay of cGMP concentration according to general principle of ELISA technique and the manufacturer's instructions (#k372-100, Biovision Research Products, USA). The kit utilizes the recombinant Protein G coated plate to anchor cGMP polyclonal antibody. cGMP-HRP conjugates directly competes with cGMP from the samples for binding to the cGMP specific antibody on the plate. After incubation and washing, the amount of cGMP-HRP bound to plate was determined by reading the optical density at 450 nm on a micorplate autoreader (Bio-Tek Instruments EL311). The absorbance is inversely proportional to the concentration of cGMP in the samples.

Determination of nitric oxide: Nitric Oxide (NO) is rapidly oxidized to nitrite and nitrate which are used to determine NO production. BioVisions Nitric Oxide Colorimetric Assay Kit (#K262-200, BioVision Research Products, USA) was used to measure the total nitrate/nitrite in the samples.

Statistical analysis: All data are presented as the Mean±SEM. Data were analysed by analysis of variance (ANOVA) followed by inspection of all differences by Tukey test for concentration of cGMP, NO and penile/body weight ratio. Mean weight were compared paired t-test. p>0.05 was considered statistically significant, statistical analysis was done using graph pad prism 5 statistical software.

Body weight of animals before and after administration of
ethanol extract of Phyllanthus amarus and penile/body weight
ratio after administration

Value are Mean±SD. Values with same superscript are
not significant within columns for penile/body weight ratio at p>0.05.
Paired comparism was done for body weight

The mean weight difference of animals at day zero (before administration) and
day ten (after administration) of plant extract and controls was not significant
(p>0.05) (Table 2).

There was a significant difference (p<0.05) when the penile/body weight ratio of animals in groups 1 (saline), 3 (100 mg kg-1) and 4 (200 mg kg-1) were compared to groups 2 (Sildenafil) and 5 (400 mg kg-1) but there was no difference (p>0.05) when group 1 was compared with groups 3 and 4 (Table 2).

Figure 1 shows a dose dependent increase in the concentration of cGMP in groups administered the ethanol plant extract. The statistical difference in the concentration of cGMP in animals administered lower doses (100 and 200 mg kg-1) of the plant extract was not significant when compared with those administered physiological saline but significant compared with those administered sildenafil. Also, the difference in the level of cGMP was significant in the groups administered the lower doses and physiological saline when compared to the group administered the highest dose (400 mg kg-1). However, the difference between the 400 mg kg-1 group was significant compared to the sildenafil group.

The concentration of Nitric Oxide (NO) though highest in group 1, showed no significant difference when compared with all the other four groups. Also there was no significant difference when the level of NO was compared within groups.

The use of herbs is very common in developing countries, particularly in rural
settings. However, during the last decade an increase in the use of plants has
been observed in metropolitan areas of developed countries (Harnack
et al., 2001).

Sexual difficulties are extremely prevalent among both men and women. They
are associated with a number of biological, medical and psychological risk factors
and increase markedly with aging (Leiblum, 1999). The
phytochemicals present in Phyllanthus amarus include: flavonoids, tannins,
saponins, alkaloids, terpenoids, steroids and cardiac glycosides (Table
1). Flavonoids have been shown to possess many pharmacological properties
such as anti-oxidant activities, anti-inflammatory activities, anti- cancer
activities and anti- microbial effects hence, flavonoids may have a contributory
effect to the fertility properties and other pharmacological effects of the
plant (Adeneye et al., 2006).

Etta (2008) reported a decrease in litter size and
weight of albino rats intraperitoneally administered the ethanolic extract of
the whole plant. This is in contrast with the result gotten from this research,
which showed no significant difference in the weights of animals after 10 days
of administration of the plant extract, (p>0.05) for all the treatment groups
(Table 2). This is an indication that the ethanolic plant
extract does not have any adverse effect on the weight of adult animals.

Improving sexual desire and function is possible using plant-based medicines
that improve the activity of the male glandular system, improve the blood supply
to erectile tissue and enhance the transmission or stimulation of the nerve
signal. A number of natural agents occur to boost sexual function and could
be appropriately described also as aphrodisiacs. Table 2 shows
the body weight per penile weight ratio after administration of the plant extract.
There was no significant difference in the penile weight per body weight ratio
in animals administered physiological saline and those administered lower doses
of the plant extract (100 and 200 mg kg-1) but when compared with
group2 (sildenafil) and group5 (400 mg kg-1), the difference in body
weight per penile weight ratio of animals in group1 was significant. Also, the
body weight per penile weight ratio of animals in groups 3 and 4 were significant
when compared to those in groups 2 and 4 (p<0.05). Preoccupation with penile
size is a common condition in the male population. Penile length, circumference
and/or geometry may also contribute to, or be a consequence of, medical treatment
of erectile dysfunction (Shamloul, 2005). A higher penile/body
weight ratio is an indication of the plant extract being a treatment for erectile
dysfunction.

The discovery that penile erection is dependent on nitrergic neurons and subject
to modulation by zaprinast (Rajfer et al., 1992)
promoted the development of selective PDE 5 inhibitors. At present selective
inhibitors of PDE 5 (Sildenafil, Tadenafil and Verdenafil) is use for treatment
of male erectile dysfunction and they do this by increasing cellular content
of cGMP (Eardley and Cartledge, 2002; Francis
and Corbin, 2003). Sildenafil citrate is a useful drug for the treatment
of Erectile Dysfunction (ED) because it selectively inhibits phosphodiesterase
type 5 (PDE-5) which inactivates cyclic guanosine monophosphate (cGMP), the
mediator of smooth muscle relaxation in the corpus cavernosum. By selectively
inhibiting cGMP catabolism in cavernosal smooth-muscle cells, sildenafil citrate
can restore the natural erectile response to sexual stimulation without causing
erections in the absence of such stimulation. Sildenafil citrate is rapidly
absorbed, with maximal plasma concentrations occurring within 1hr after oral
administration and a mean terminal half-life of 3 to 5 h (Goldstein,
1988).

There was a dose dependent increase in the concentration of cGMP in the three study groups fed with the extract. The levels of cGMP in animals in groups 3 and 4 though higher compared to the levels in group1, the difference was not significant (p>0.05). The cGMP concentration in the control (group 1) was significantly different (p<0.05) when compared with groups 2 and 5. Also the difference in the concentration of cGMP in animals fed with lower doses of P. amarus extract (groups 3 and 4) was significant when compared with groups 2 and 5. There was a significant difference in the cGMP concentration between the sildenafil group and 400 mg kg-1 body weight group (group 5) (Fig. 1). This might be an indication that the extract has an effect directly or indirectly on muscle cGMP but the effect could only be noticed at higher concentration.

Hallen et al. (2001) reported that by quantifying
the neuronal release of Nitric oxide from guinea pig colon and rabbit corpus
cavernosum, the selective PDE 5 inhibitors altered the nerve-induced release
of NO. On the other hand, Ibrahim et al. (2004),
concluded that the PDE 5 inhibitor zaprinast has no regulatory effect on the
NO-release in serum and aortic tissue though the mean arterial pressure was
lowered.

The result showed the ethanolic plant extract did not have effect on the level
of Nitric Oxide (NO). Although the concentration of NO was lower compared to
the control, the differences were not significant (Fig. 2).
PDE 5 inhibitors have been reported to have a great tendency to regulate the
level of nitric oxide through its inhibitory action on nitric oxide synthase
(NOs). Application of sildenafil, tadalafil and verdenafil all decreased the
nerve-induced release of NO. This is an indication that PDE 5 influences the
formation and release of NO during stimulation in the cavernous tissue.

It was suggested that accumulated cGMP reduces NO by phosphorylating nNOS
by protein kinase A or protein kinase G (Dinerman et
al., 1994). Though present result shows no significant difference in
concentration of NO in studied groups compared to the control, the concentration
was stilled reduced by the plant extract. This is an indication that higher
concentration of the extract might lead to accumulation of cGMP which will lead
to reduction of NO.

The diminished release of NO suspected to be mediated by the plant extract
might likely be of significant physiological and pharmacological importance.
Burnett (2003) reported that if selective PDE 5 inhibitors
increases endogenously formed NO, it would start a positive feedback circle,
which might ultimately have induced priapism. However since the selective PDE
5 inhibitors diminished the formation of NO, NO on its own formation prevents
priapism through negative feedback either via cGMP-dependent phosphorylation
of NOs or via NO-inhibition of NOs.

CONCLUSION

Ethanol extracts of Phyllanthus amarus may have positive penile erection property as shown by its ability to increase the cGMP level in penile tissue of male guinea pigs. The increase in level of cGMP is dose dependent and the action of ethanol extract of the plant is probably through the inhibition of phosphodiesterase 5 an enzyme known to break down cGMP in corpus cavernosum in male penile tissue. The ethanol extract of the plant shows no significant effect on the level of plasma nitric oxide.

However, more research need to be carried out in confirming the plant as a source of a phosphodiesterase inhibitor, isolating the actual active ingredient (s) responsible for this action and determining its actual effect on nitric oxide.